Microrna expression in human peripheral blood microvesicles and uses thereof

ABSTRACT

The present invention provides novel methods and compositions for the diagnosis, prognosis and treatment of disorders by examining samples containing microvesicles and miRs therein.

This application is a continuation of U.S. application Ser. No.13/873,371 filed Apr. 30, 2013, which application is a continuation ofU.S. application Ser. No. 12/677,931 filed Apr. 12, 2010, now U.S. Pat.No. 8,455,199 issued Jun. 4, 2013, which claims the benefit of PCTapplication No. PCT/US08/076109 filed Sep. 12, 2008 which claimspriority to U.S. Provisional Patent Application 60/993,809 filed Sep.14, 2007, and U.S. Provisional Patent Application 61/055,178 filed May22, 2008, all of which applications are fully incorporated herein byreference. This invention was not made with any government support andthe government has no rights in this invention.

SEQUENCE LISTING

The Instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created Sep. 23, 2016, isnamed “37901792302.txt” and is 43,469 bytes in size.

BACKGROUND OF THE INVENTION

MicroRNAs (miRNAs or miRs) are small non-coding RNAs expressed inanimals and plants. They regulate cellular function, cell survival, cellactivation and cell differentiation during development.^(7;8)

MicroRNAs are a small non-coding family of 19-25 nucleotide RNAs thatregulate gene expression by targeting messenger RNAs (mRNA) in asequence specific manner, inducing translational repression or mRNAdegradation depending on the degree of complementarity between miRNAsand their targets (Bartel, D. P. (2004) Cell 116, 281-297; Ambros, V.(2004) Nature 431, 350-355). Many miRs are conserved in sequence betweendistantly related organisms, suggesting that these molecules participatein essential processes. Indeed, miRs are involved in the regulation ofgene expression during development (Xu, P., et al. (2003) Curr. Biol.13, 790-795), cell proliferation (Xu, P., et al. (2003) Curr. Biol. 13,790-795), apoptosis (Cheng, A. M., et al. (2005) Nucl. Acids Res. 33,1290-1297), glucose metabolism (Poy, M. N., et al. (2004) Nature 432,226-230), stress resistance (Dresios, J., et al. (2005) Proc. Natl.Acad. Sci. USA 102, 1865-1870) and cancer (Calin, G. A, et al. (2002)Proc. Natl. Acad. Sci. USA 99, 1554-15529; Calin, G. A., et al. (2004)Proc. Natl. Acad. Sci. USA 101, 11755-11760; He, L., et al. (2005)Nature 435, 828-833; and Lu, J., et al. (2005) Nature 435:834-838).

There is also strong evidence that miRs play a role in mammalianhematopoiesis. In mice, miR-181, miR-223 and miR-142 are differentiallyexpressed in hematopoietic tissues, and their expression is regulatedduring hematopoiesis and lineage commitment (Chen, C. Z., et al. (2004)Science 303, 83-86). The ectopic expression of miR-181 in murinehematopoietic progenitor cells led to proliferation in the B-cellcompartment (Chen, C. Z., et al. (2004) Science 303, 83-86). SystematicmiR gene profiling in cells of the murine hematopoietic system revealeddifferent miR expression patterns in the hematopoietic system comparedwith neuronal tissues, and identified individual miR expression changesthat occur during cell differentiation (Monticelli, S., et al. (2005)Genome Biology 6, R71). A recent study has identified down-modulation ofmiR-221 and miR-222 in human erythropoietic cultures of CD34⁺ cord bloodprogenitor cells (Felli, N., et al. (2005) Proc. Natl. Acad. Sci. USA.102, 18081-18086). These miRs were found to target the oncogene c-Kit.Further functional studies indicated that the decline of these two miRsin erythropoietic cultures unblocks Kit protein production at thetranslational level leading to expansion of early erythroid cells(Felli, N., et al. (2005) Proc. Natl. Acad. Sci. USA. 102, 18081-18086).In line with the hypothesis of miRs regulating cell differentiation,miR-223 was found to be a key member of a regulatory circuit involvingC/EBPa and NFI-A, which controls granulocytic differentiation inall-trans retinoic acid-treated acute promyelocytic leukemic cell lines(Fazi, F., et al. (2005) Cell 123, 819-831).

A frequent deletion and reduced expression of two miRs in B-cell chroniclymphocytic leukemia has been identified⁹. This discovery stimulatednumerous articles documenting aberrant expression of miRs in head andneck carcinomas, small cell lung cancers, glioblastomas, breast cancers,chronic lymphocytic leukemia, and Burkitt lymphoma.⁹⁻¹² More recently, arelationship between inflammation and miRs has been reported inmacrophages.¹³

In order to test for such disorders, tissue samples have been obtainedin order to confirm the presence of such macrophages. In addition, untilnow, there has been no report demonstrating that microvesicles thatcirculate in the blood contain miRs.

Additional advantages, objects, and features of the invention will beset forth in part in the description which follows and in part willbecome apparent to those having ordinary skill in the art uponexamination of the following or may be learned from practice of theinvention. The objects and advantages of the invention may be realizedand attained as particularly pointed out in the appended claims.

SUMMARY OF THE INVENTION

In one aspect, there is provided a method for identifying specific miRsthat are present in microvesicles and/or have altered expression levelsof specific miRs in tissue, fluids and/or cells.

Microvesicles facilitate communication between cells. Many cellsincluding macrophages, platelets, T-cells, and tumors release smallmicrovesicles containing nucleic acids and/or proteins¹⁻⁵. Factorscontained within the microvesicles regulate angiogenesis, cell growth,and cell differentiation^(1;3).

In another aspect, the presence of miRs in such fluids as peripheralblood of patients suffering from particular disorders is determined.

In another aspect, the presence of miRs in lung tissue of patientssuffering from pulmonary fibrosis is determined

In yet another aspect, there is provided herein a method of diagnosingor prognosticating a particular disorder in a subject (e.g., a human)According to one particular method, the level of at least one miR geneproduct in a test sample from the subject is compared to the level of acorresponding miR gene product in a control sample. An alteration (e.g.,an increase, a decrease) in the level of the miR gene product in thetest sample, relative to the level of a corresponding miR gene productin the control sample, is indicative of the subject either having, orbeing at risk for developing, an acute inflammatory disorder.

In one embodiment, the level of the miR gene product in the test samplefrom the subject is greater than that of the control. In anotherembodiment, the at least one miR gene product is selected from the groupconsisting of the miRNAs as shown herein.

In particular embodiments, the disorder that is diagnosed orprognosticated is one that causes mononuclear phagocytes and/or THP-1cells to release microvesicles.

In particular embodiments, the disorder that is diagnosed orprognosticated is one that causes an inflammatory response.

In another embodiment, the invention is a method of treating a cancerand/or an inflammatory disorder in a subject (e.g., a human).

In one particular method, an effective amount of a compound forinhibiting expression of at least one miR gene product selected from theone or more of the groups found in Table I-VI is administered to thesubject.

In one embodiment, the compound for inhibiting expression of at leastone miR gene product inhibits expression of a miR gene product selectedfrom one or more of the groups found in Tables I-VI.

The invention further provides pharmaceutical compositions for treatingcancer and/or an inflammatory disorder. In one embodiment, thepharmaceutical compositions of the invention comprise at least one miRexpression-inhibition compound and a pharmaceutically-acceptablecarrier. In a particular embodiment, the at least one miRexpression-inhibition compound is specific for a miR gene product whoseexpression is greater in blood from diseased patients compared tonormals.

In yet another embodiment, the pharmaceutical composition furthercomprises at least one anti-inflammatory agent.

In one embodiment, the invention is a pharmaceutical composition fortreating a cancer associated with overexpression of a miR gene productand/or a lung disorder associated with overexpression of a miR geneproduct. Such pharmaceutical compositions comprise an effective amountof at least one miR gene product and a pharmaceutically-acceptablecarrier, wherein the at least one miR gene product binds to, anddecreases expression of, the miR gene product. In another embodiment,the at least one miR gene product comprises a nucleotide sequence thatis complementary to a nucleotide sequence in the miR- gene product. Instill another embodiment, the at least one miR gene product is miR- or avariant or biologically-active fragment thereof. In yet anotherembodiment, the pharmaceutical composition further comprises at leastone anti-cancer agent.

Various objects and advantages of this invention will become apparent tothose skilled in the art from the following detailed description of thepreferred embodiment, when read in light of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the differentiation induced release of microvesicles frommacrophages. Peripheral blood monocytes (PBM) were untreated (light) ortreated with GM-CSF (dark) for 24 h. Cell-free supernatant was collectedand ultracentrifuged. The vesicles were resuspended in PBS and analyzedfor size on a flow cytometry. Prior to analysis, FSS and SSC parameterswere adjusted using 2 μm standard beads (not shown). Shown isrepresentative data from three different donors.

FIGS. 2A-2C show microvesicles mediate macrophage differentiation.Microvesicles were collected from PMA-treated THP1 cells then added toundifferentiated THP1 cells (FIG. 2B) or monocytes (FIG. 2C). As acontrol, THP1 cells were left untreated (FIG. 2A). The cells werephotographed daily. Shown are the cells at day 3.

FIGS. 3A-3C show the isolation of peripheral blood microvesicles.Following informed consent, plasma was obtained from 20 cc of blood fromnormal volunteer donors. The microvesicles from 0.5 cc of plasma wereincubated with CD206-FITC or MHCII-FITC antibodies and analyzed on BDFACS Calibur for size using forward vs. side scatter (FIG. 3A) andsurface antigen expression (FIG. 3B). The percent expression of eitherCD206 or MHC II compared to isotype control was determined for the gatedregion shown in FIG. 3A (FIG. 3C). Shown is the average±SEM of twodonors.

FIG. 4. Analysis of the origin of peripheral blood microvesicles.Peripheral blood microvesicles from healthy donors (n=10) were analyzedby flow cytometry. To determine cell origin, microvesicles were stainedfor CD3, CD202b (Tie-2), CD66b, CD79a, or CD41a to determine those thatoriginated from T-cells, endothelial cells, neutrophils, B-cells, orplatelets. Mononuclear phagocyte-derived microvesicles were positive forCD14, CD206, CCR3, CCR2, or CCRS. Shown is the average % maximum oftotal gated events ±S.E.M.

FIGS. 5A-5D. miRNA expression from peripheral blood microvesicles andPBMC. (FIG. 5A) Hierarchal cluster analysis for microvesicles and PBMCis shown based on filtering criteria. Heat-maps demonstrating theexpression profile for microvesicles (FIG. 5B) and PBMC (FIG. 5C) weregenerated. (FIG. 5D). The number of shared and specific for each samplegroup is shown.

FIG. 6: Table I showing various diseases and up- and down-regulated miRsassociated therewith. microRNAs that are important in tissue of humandiseases, including cancer and non-cancer applications are listed.Comparing miRNAs that are undetectable in the plasma from our data set(FIG. 7, Table II) with miRNAs known to increase in the tissue ofspecific diseases , the inventors now believe that we predict thatseveral miRNAs may serve as biomarkers in the plasma (see miRs in boldin FIG. 6, Table I Increase Expression Column).

FIG. 7: Table II showing miRs that are expressed in the plasma and thosethat are undetectable.

FIG. 8: Table III lists miRs and show the top ten expressed miRNAs inthe plasma microvesicles and the PBMC from all individuals.

FIG. 9: Table IV showing canonical pathways involved in metabolism andregulation of the acquired immune system were highly regulated by theexpression of these miRNAs using Sanger miRBase alone (top) or commontargets from Sanger miRBase and TargetScan (bottom).

FIG. 10: Table V showing that 20 miRNAs had more than a three-foldincrease in expression in the PBMC fraction compared to themicrovesicles plasma samples as well as the fold change in plasmamicrovesicles compared to PMBC (last Column)

FIG. 11: Table VI showing, the normalized expression data for alldetected miRs: detector name, ave-MNC, std-MNC, detector name,ave-serum, std-serum.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is based, in part, on the identification ofspecific microRNAs (miRNAs) that are involved in an inflammatoryresponse and/or have altered expression levels in blood. The inventionis further based, in part, on association of these miRNAs withparticular diagnostic, prognostic and therapeutic features.

As described and exemplified herein particular miRNA are up- ordown-regulated during tissue injury and/or inflammation.

As used herein interchangeably, a “miR gene product,” “microRNA,” “miR,”“miR” or “miRNA” refers to the unprocessed or processed RNA transcriptfrom a miR gene. As the miR gene products are not translated intoprotein, the term “miR gene products” does not include proteins. Theunprocessed miR gene transcript is also called a “miR precursor,” andtypically comprises an RNA transcript of about 70-100 nucleotides inlength. The miR precursor can be processed by digestion with an RNAse(for example, Dicer, Argonaut, RNAse III (e.g., E. coli RNAse III)) intoan active 19-25 nucleotide RNA molecule. This active 19-25 nucleotideRNA molecule is also called the “processed” miR gene transcript or“mature” miRNA.

The active 19-25 nucleotide RNA molecule can be obtained from the miRprecursor through natural processing routes (e.g., using intact cells orcell lysates) or by synthetic processing routes (e.g., using isolatedprocessing enzymes, such as isolated Dicer, Argonaut, or RNAse III). Itis understood that the active 19-25 nucleotide RNA molecule can also beproduced directly by biological or chemical synthesis, without having tobe processed from the miR precursor. When a microRNA is referred toherein by name, the name corresponds to both the precursor and matureforms, unless otherwise indicated.

The present invention encompasses methods of diagnosing orprognosticating whether a subject has, or is at risk for developing, adisorder where microvesicles are released.

The methods comprise determining the level of at least one miR geneproduct in a sample from the subject and comparing the level of the miRgene product in the sample to a control. As used herein, a “subject” canbe any mammal that has, or is suspected of having, such disorder. In apreferred embodiment, the subject is a human who has, or is suspected ofhaving, such disorder.

The level of at least one miR gene product can be measured in cells of abiological sample obtained from the subject.

In another embodiment, a sample can be removed from the subject, and DNAcan be extracted and isolated by standard techniques. For example, incertain embodiments, the sample can be obtained from the subject priorto initiation of radiotherapy, chemotherapy or other therapeutictreatment. A corresponding control sample, or a control reference sample(e.g., obtained from a population of control samples), can be obtainedfrom unaffected samples of the subject, from a normal human individualor population of normal individuals, or from cultured cellscorresponding to the majority of cells in the subject's sample. Thecontrol sample can then be processed along with the sample from thesubject, so that the levels of miR gene product produced from a givenmiR gene in cells from the subject's sample can be compared to thecorresponding miR gene product levels from cells of the control sample.Alternatively, a reference sample can be obtained and processedseparately (e.g., at a different time) from the test sample and thelevel of a miR gene product produced from a given miR gene in cells fromthe test sample can be compared to the corresponding miR gene productlevel from the reference sample.

In one embodiment, the level of the at least one miR gene product in thetest sample is greater than the level of the corresponding miR geneproduct in the control sample (i.e., expression of the miR gene productis “upregulated”). As used herein, expression of a miR gene product is“upregulated” when the amount of miR gene product in a sample from asubject is greater than the amount of the same gene product in a control(for example, a reference standard, a control cell sample, a controltissue sample).

In another embodiment, the level of the at least one miR gene product inthe test sample is less than the level of the corresponding miR geneproduct in the control sample (i.e., expression of the miR gene productis “downregulated”). As used herein, expression of a miR gene is“downregulated” when the amount of miR gene product produced from thatgene in a sample from a subject is less than the amount produced fromthe same gene in a control sample. The relative miR gene expression inthe control and normal samples can be determined with respect to one ormore RNA expression standards. The standards can comprise, for example,a zero miR gene expression level, the miR gene expression level in astandard cell line, the miR gene expression level in unaffected samplesof the subject, or the average level of miR gene expression previouslyobtained for a population of normal human controls (e.g., a controlreference standard).

The level of the at least one miR gene product can be measured using avariety of techniques that are well known to those of skill in the art(e.g., quantitative or semi-quantitative RT-PCR, Northern blot analysis,solution hybridization detection). In a particular embodiment, the levelof at least one miR gene product is measured by reverse transcribing RNAfrom a test sample obtained from the subject to provide a set of targetoligodeoxynucleotides, hybridizing the target oligodeoxynucleotides toone or more miRNA-specific probe oligonucleotides (e.g., a microarraythat comprises miRNA-specific probe oligonucleotides) to provide ahybridization profile for the test sample, and comparing the test samplehybridization profile to a hybridization profile generated from acontrol sample. An alteration in the signal of at least one miRNA in thetest sample relative to the control sample is indicative of the subjecteither having, or being at risk for a particular disorder.

Also, a microarray can be prepared from gene-specific oligonucleotideprobes generated from known miRNA sequences. The array may contain twodifferent oligonucleotide probes for each miRNA, one containing theactive, mature sequence and the other being specific for the precursorof the miRNA. The array may also contain controls, such as one or moremouse sequences differing from human orthologs by only a few bases,which can serve as controls for hybridization stringency conditions.tRNAs and other RNAs (e.g., rRNAs, mRNAs) from both species may also beprinted on the microchip, providing an internal, relatively stable,positive control for specific hybridization. One or more appropriatecontrols for non-specific hybridization may also be included on themicrochip. For this purpose, sequences are selected based upon theabsence of any homology with any known miRNAs.

The microarray may be fabricated using techniques known in the art. Forexample, probe oligonucleotides of an appropriate length, e.g., 40nucleotides, are 5′-amine modified at position C6 and printed usingcommercially available microarray systems, e.g., the GeneMachineOmniGrid™ 100 Microarrayer and Amersham CodeLink™ activated slides.Labeled cDNA oligomer corresponding to the target RNAs is prepared byreverse transcribing the target RNA with labeled primer. Following firststrand synthesis, the RNA/DNA hybrids are denatured to degrade the RNAtemplates. The labeled target cDNAs thus prepared are then hybridized tothe microarray chip under hybridizing conditions, e.g., 6× SSPE/30%formamide at 25° C. for 18 hours, followed by washing in 0.75×TNT at 37°C. for 40 minutes. At positions on the array where the immobilized probeDNA recognizes a complementary target cDNA in the sample, hybridizationoccurs. The labeled target cDNA marks the exact position on the arraywhere binding occurs, allowing automatic detection and quantification.The output consists of a list of hybridization events, indicating therelative abundance of specific cDNA sequences, and therefore therelative abundance of the corresponding complementary miRs, in thepatient sample. According to one embodiment, the labeled cDNA oligomeris a biotin-labeled cDNA, prepared from a biotin-labeled primer. Themicroarray is then processed by direct detection of thebiotin-containing transcripts using, e.g., Streptavidin-Alexa647conjugate, and scanned utilizing conventional scanning methods. Imageintensities of each spot on the array are proportional to the abundanceof the corresponding miR in the patient sample.

The use of the array has several advantages for miRNA expressiondetection. First, the global expression of several hundred genes can beidentified in the same sample at one time point. Second, through carefuldesign of the oligonucleotide probes, expression of both mature andprecursor molecules can be identified. Third, in comparison withNorthern blot analysis, the chip requires a small amount of RNA, andprovides reproducible results using 2.5 μg of total RNA. The relativelylimited number of miRNAs (a few hundred per species) allows theconstruction of a common microarray for several species, with distinctoligonucleotide probes for each. Such a tool allows for analysis oftrans-species expression for each known miR under various conditions.

In addition to use for quantitative expression level assays of specificmiRs, a microchip containing miRNA-specific probe oligonucleotidescorresponding to a substantial portion of the miRNome, preferably theentire miRNome, may be employed to carry out miR gene expressionprofiling, for analysis of miR expression patterns. Distinct miRsignatures can be associated with established disease markers, ordirectly with a disease state.

According to the expression profiling methods described herein, totalRNA from a sample from a subject suspected of having a particulardisorder is quantitatively reverse transcribed to provide a set oflabeled target oligodeoxynucleotides complementary to the RNA in thesample. The target oligodeoxynucleotides are then hybridized to amicroarray comprising miRNA-specific probe oligonucleotides to provide ahybridization profile for the sample. The result is a hybridizationprofile for the sample representing the expression pattern of miRNA inthe sample. The hybridization profile comprises the signal from thebinding of the target oligodeoxynucleotides from the sample to themiRNA-specific probe oligonucleotides in the microarray. The profile maybe recorded as the presence or absence of binding (signal vs. zerosignal). More preferably, the profile recorded includes the intensity ofthe signal from each hybridization. The profile is compared to thehybridization profile generated from a normal control sample orreference sample. An alteration in the signal is indicative of thepresence of, or propensity to develop, the particular disorder in thesubject.

Other techniques for measuring miR gene expression are also within theskill in the art, and include various techniques for measuring rates ofRNA transcription and degradation.

The invention also provides methods of diagnosing whether a subject has,or is at risk for developing, a particular disorder with an adverseprognosis. In this method, the level of at least one miR gene product,which is associated with an adverse prognosis in a particular disorder,is measured by reverse transcribing RNA from a test sample obtained fromthe subject to provide a set of target oligodeoxynucleotides. The targetoligodeoxynucleotides are then hybridized to one or more miRNA-specificprobe oligonucleotides (e.g., a microarray that comprises miRNA-specificprobe oligonucleotides) to provide a hybridization profile for the testsample, and the test sample hybridization profile is compared to ahybridization profile generated from a control sample. An alteration inthe signal of at least one miRNA in the test sample relative to thecontrol sample is indicative of the subject either having, or being atrisk for developing, a particular disorder with an adverse prognosis.

In some instances, it may be desirable to simultaneously determine theexpression level of a plurality of different miR gene products in asample. In other instances, it may be desirable to determine theexpression level of the transcripts of all known miR genes correlatedwith a particular disorder. Assessing specific expression levels forhundreds of miR genes or gene products is time consuming and requires alarge amount of total RNA (e.g., at least 20 μg for each Northern blot)and autoradiographic techniques that require radioactive isotopes.

To overcome these limitations, an oligolibrary, in microchip format(i.e., a microarray), may be constructed containing a set ofoligonucleotide (e.g., oligodeoxynucleotide) probes that are specificfor a set of miR genes. Using such a microarray, the expression level ofmultiple microRNAs in a biological sample can be determined by reversetranscribing the RNAs to generate a set of target oligodeoxynucleotides,and hybridizing them to probe the oligonucleotides on the microarray togenerate a hybridization, or expression, profile. The hybridizationprofile of the test sample can then be compared to that of a controlsample to determine which microRNAs have an altered expression level. Asused herein, “probe oligonucleotide” or “probe oligodeoxynucleotide”refers to an oligonucleotide that is capable of hybridizing to a targetoligonucleotide. “Target oligonucleotide” or “targetoligodeoxynucleotide” refers to a molecule to be detected (e.g., viahybridization). By “miR-specific probe oligonucleotide” or “probeoligonucleotide specific for a miR” is meant a probe oligonucleotidethat has a sequence selected to hybridize to a specific miR geneproduct, or to a reverse transcript of the specific miR gene product.

An “expression profile” or “hybridization profile” of a particularsample is essentially a fingerprint of the state of the sample; whiletwo states may have any particular gene similarly expressed, theevaluation of a number of genes simultaneously allows the generation ofa gene expression profile that is unique to the state of the cell. Thatis, normal samples may be distinguished from correspondingdisorder-exhibiting samples. Within such disorder-exhibiting samples,different prognosis states (for example, good or poor long term survivalprospects) may be determined By comparing expression profiles ofdisorder-exhibiting samples in different states, information regardingwhich genes are important (including both upregulation anddownregulation of genes) in each of these states is obtained.

The identification of sequences that are differentially expressed indisorder-exhibiting samples, as well as differential expressionresulting in different prognostic outcomes, allows the use of thisinformation in a number of ways. For example, a particular treatmentregime may be evaluated (e.g., to determine whether a chemotherapeuticdrug acts to improve the long-term prognosis in a particular subject).Similarly, diagnosis may be done or confirmed by comparing samples froma subject with known expression profiles. Furthermore, these geneexpression profiles (or individual genes) allow screening of drugcandidates that suppress the particular disorder expression profile orconvert a poor prognosis profile to a better prognosis profile.

Alterations in the level of one or more miR gene products in cells canresult in the deregulation of one or more intended targets for thesemiRs, which can lead to a particular disorder. Therefore, altering thelevel of the miR gene product (e.g., by decreasing the level of a miRthat is upregulated in disorder-exhibiting cells, by increasing thelevel of a miR that is downregulated in disorder-exhibiting cells) maysuccessfully treat the disorder.

Accordingly, the present invention encompasses methods of treating adisorder in a subject, wherein at least one miR gene product isderegulated (e.g., downregulated, upregulated) in the cells of thesubject. In one embodiment, the level of at least one miR gene productin a test sample is greater than the level of the corresponding miR geneproduct in a control or reference sample. In another embodiment, thelevel of at least one miR gene product in a test sample is less than thelevel of the corresponding miR gene product in a control sample. Whenthe at least one isolated miR gene product is downregulated in the testsample, the method comprises administering an effective amount of the atleast one isolated miR gene product, or an isolated variant orbiologically-active fragment thereof, such that proliferation of thedisorder-exhibiting cells in the subject is inhibited.

For example, when a miR gene product is downregulated in a cancer cellin a subject, administering an effective amount of an isolated miR geneproduct to the subject can inhibit proliferation of the cancer cell. Theisolated miR gene product that is administered to the subject can beidentical to an endogenous wild-type miR gene product that isdownregulated in the cancer cell or it can be a variant orbiologically-active fragment thereof.

As defined herein, a “variant” of a miR gene product refers to a miRNAthat has less than 100% identity to a corresponding wild-type miR geneproduct and possesses one or more biological activities of thecorresponding wild-type miR gene product. Examples of such biologicalactivities include, but are not limited to, inhibition of expression ofa target RNA molecule (e g , inhibiting translation of a target RNAmolecule, modulating the stability of a target RNA molecule, inhibitingprocessing of a target RNA molecule) and inhibition of a cellularprocess associated with cancer and/or a myeloproliferative disorder(e.g., cell differentiation, cell growth, cell death). These variantsinclude species variants and variants that are the consequence of one ormore mutations (e.g., a substitution, a deletion, an insertion) in a miRgene. In certain embodiments, the variant is at least about 70%, 75%,80%, 85%, 90%, 95%, 98%, or 99% identical to a corresponding wild-typemiR gene product.

As defined herein, a “biologically-active fragment” of a miR geneproduct refers to an RNA fragment of a miR gene product that possessesone or more biological activities of a corresponding wild-type miR geneproduct. As described above, examples of such biological activitiesinclude, but are not limited to, inhibition of expression of a targetRNA molecule and inhibition of a cellular process associated with cancerand/or a myeloproliferative disorder. In certain embodiments, thebiologically-active fragment is at least about 5, 7, 10, 12, 15, or 17nucleotides in length. In a particular embodiment, an isolated miR geneproduct can be administered to a subject in combination with one or moreadditional anti-cancer treatments. Suitable anti-cancer treatmentsinclude, but are not limited to, chemotherapy, radiation therapy andcombinations thereof (e.g., chemoradiation).

When the at least one isolated miR gene product is upregulated in thecancer cells, the method comprises administering to the subject aneffective amount of a compound that inhibits expression of the at leastone miR gene product, such that proliferation of the disorder-exhibitingcells is inhibited. Such compounds are referred to herein as miR geneexpression-inhibition compounds. Examples of suitable miR geneexpression-inhibition compounds include, but are not limited to, thosedescribed herein (e.g., double-stranded RNA, antisense nucleic acids andenzymatic RNA molecules).

In a particular embodiment, a miR gene expression-inhibiting compoundcan be administered to a subject in combination with one or moreadditional anti-cancer treatments. Suitable anti-cancer treatmentsinclude, but are not limited to, chemotherapy, radiation therapy andcombinations thereof (e.g., chemoradiation).

As described herein, when the at least one isolated miR gene product isupregulated in cancer cells, the method comprises administering to thesubject an effective amount of at least one compound for inhibitingexpression of the at least one miR gene product, such that proliferationof cancer cells is inhibited.

The terms “treat”, “treating” and “treatment”, as used herein, refer toameliorating symptoms associated with a disease or condition, forexample, cancer and/or other condition or disorder, including preventingor delaying the onset of the disease symptoms, and/or lessening theseverity or frequency of symptoms of the disease, disorder or condition.The terms “subject”, “patient” and “individual” are defined herein toinclude animals, such as mammals, including, but not limited to,primates, cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs,rats, mice or other bovine, ovine, equine, canine, feline, rodent, ormurine species. In a preferred embodiment, the animal is a human

As used herein, an “isolated” miR gene product is one that issynthesized, or altered or removed from the natural state through humanintervention. For example, a synthetic miR gene product, or a miR geneproduct partially or completely separated from the coexisting materialsof its natural state, is considered to be “isolated.” An isolated miRgene product can exist in a substantially-purified form, or can exist ina cell into which the miR gene product has been delivered. Thus, a miRgene product that is deliberately delivered to, or expressed in, a cellis considered an “isolated” miR gene product. A miR gene productproduced inside a cell from a miR precursor molecule is also consideredto be an “isolated” molecule. According to the invention, the isolatedmiR gene products described herein can be used for the manufacture of amedicament for treating a subject (e.g., a human)

Isolated miR gene products can be obtained using a number of standardtechniques. For example, the miR gene products can be chemicallysynthesized or recombinantly produced using methods known in the art. Inone embodiment, miR gene products are chemically synthesized usingappropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesizer. Commercial suppliers of synthetic RNAmolecules or synthesis reagents include, e.g., Proligo (Hamburg,Germany), Dharmacon Research (Lafayette, Colo., U.S.A.), Pierce Chemical(part of Perbio Science, Rockford, Ill., U.S.A.), Glen Research(Sterling, Va., U.S.A.), ChemGenes (Ashland, Mass., U.S.A.) and Cruachem(Glasgow, UK).

Alternatively, the miR gene products can be expressed from recombinantcircular or linear DNA plasmids using any suitable promoter. Suitablepromoters for expressing RNA from a plasmid include, e.g., the U6 or H1RNA pol III promoter sequences, or the cytomegalovirus promoters.Selection of other suitable promoters is within the skill in the art.The recombinant plasmids of the invention can also comprise inducible orregulatable promoters for expression of the miR gene products in cells(e.g., cancerous cells, cells exhibiting a myeloproliferative disorder).

The miR gene products that are expressed from recombinant plasmids canbe isolated from cultured cell expression systems by standardtechniques. The miR gene products that are expressed from recombinantplasmids can also be delivered to, and expressed directly in, cells.

The miR gene products can be expressed from a separate recombinantplasmid, or they can be expressed from the same recombinant plasmid. Inone embodiment, the miR gene products are expressed as RNA precursormolecules from a single plasmid, and the precursor molecules areprocessed into the functional miR gene product by a suitable processingsystem, including, but not limited to, processing systems extant withina cancer cell.

Selection of plasmids suitable for expressing the miR gene products,methods for inserting nucleic acid sequences into the plasmid to expressthe gene products, and methods of delivering the recombinant plasmid tothe cells of interest are within the skill in the art. See, for example,Zeng et al. (2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat.Biotechnol, 20:446-448; Brummelkamp et al. (2002), Science 296:550-553;Miyagishi et al. (2002), Nat. Biotechnol. 20:497-500; Paddison et al.(2002), Genes Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol.20:500-505; and Paul et al. (2002), Nat. Biotechnol. 20:505-508, theentire disclosures of which are incorporated herein by reference. Forexample, in certain embodiments, a plasmid expressing the miR geneproducts can comprise a sequence encoding a miR precursor RNA under thecontrol of the CMV intermediate-early promoter. As used herein, “underthe control” of a promoter means that the nucleic acid sequencesencoding the miR gene product are located 3′ of the promoter, so thatthe promoter can initiate transcription of the miR gene product codingsequences.

The miR gene products can also be expressed from recombinant viralvectors. It is contemplated that the miR gene products can be expressedfrom two separate recombinant viral vectors, or from the same viralvector. The RNA expressed from the recombinant viral vectors can eitherbe isolated from cultured cell expression systems by standardtechniques, or can be expressed directly in cells (e.g., cancerouscells, cells exhibiting a myeloproliferative disorder).

In other embodiments of the treatment methods of the invention, aneffective amount of at least one compound that inhibits miR expressioncan be administered to the subject. As used herein, “inhibiting miRexpression” means that the production of the precursor and/or active,mature form of miR gene product after treatment is less than the amountproduced prior to treatment. One skilled in the art can readilydetermine whether miR expression has been inhibited in cells using, forexample, the techniques for determining miR transcript level discussedherein Inhibition can occur at the level of gene expression (i.e., byinhibiting transcription of a miR gene encoding the miR gene product) orat the level of processing (e.g., by inhibiting processing of a miRprecursor into a mature, active miR).

As used herein, an “effective amount” of a compound that inhibits miRexpression is an amount sufficient to inhibit proliferation of cells ina subject suffering from cancer and/or a myeloproliferative disorder.One skilled in the art can readily determine an effective amount of amiR expression-inhibiting compound to be administered to a givensubject, by taking into account factors, such as the size and weight ofthe subject; the extent of disease penetration; the age, health and sexof the subject; the route of administration; and whether theadministration is regional or systemic.

One skilled in the art can also readily determine an appropriate dosageregimen for administering a compound that inhibits miR expression to agiven subject, as described herein. Suitable compounds for inhibitingmiR gene expression include double-stranded RNA (such as short- orsmall-interfering RNA or “siRNA”), antisense nucleic acids, andenzymatic RNA molecules, such as ribozymes. Each of these compounds canbe targeted to a given miR gene product and interfere with theexpression (e.g., by inhibiting translation, by inducing cleavage and/ordegradation) of the target miR gene product.

For example, expression of a given miR gene can be inhibited by inducingRNA interference of the miR gene with an isolated double-stranded RNA(“dsRNA”) molecule which has at least 90%, for example, at least 95%, atleast 98%, at least 99%, or 100%, sequence homology with at least aportion of the miR gene product. In a particular embodiment, the dsRNAmolecule is a “short or small interfering RNA” or “siRNA.”

Administration of at least one miR gene product, or at least onecompound for inhibiting miR expression, will inhibit the proliferationof cells (e.g., cancerous cells, cells exhibiting a myeloproliferativedisorder) in a subject who has a cancer and/or a myeloproliferativedisorder. As used herein, to “inhibit the proliferation of cancerouscells or cells exhibiting a myeloproliferative disorder” means to killthe cells, or permanently or temporarily arrest or slow the growth ofthe cells Inhibition of cell proliferation can be inferred if the numberof such cells in the subject remains constant or decreases afteradministration of the miR gene products or miR geneexpression-inhibiting compounds. An inhibition of proliferation ofcancerous cells or cells exhibiting a myeloproliferative disorder canalso be inferred if the absolute number of such cells increases, but therate of tumor growth decreases.

A miR gene product or miR gene expression-inhibiting compound can alsobe administered to a subject by any suitable enteral or parenteraladministration route. Suitable enteral administration routes for thepresent methods include, e.g., oral, rectal, or intranasal delivery.Suitable parenteral administration routes include, e.g., intravascularadministration (e.g., intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature); peri- and intra-tissue injection(e.g., peri-tumoral and intra-tumoral injection, intra-retinalinjection, or subretinal injection); subcutaneous injection ordeposition, including subcutaneous infusion (such as by osmotic pumps);direct application to the tissue of interest, for example by a catheteror other placement device (e.g., a retinal pellet or a suppository or animplant comprising a porous, non-porous, or gelatinous material); andinhalation. Particularly suitable administration routes are injection,infusion and direct injection into the tumor.

The miR gene products or miR gene expression-inhibition compounds can beformulated as pharmaceutical compositions, sometimes called“medicaments,” prior to administering them to a subject, according totechniques known in the art. Accordingly, the invention encompassespharmaceutical compositions for treating cancer and/or amyeloproliferative disorder.

The present pharmaceutical compositions comprise at least one miR geneproduct or miR gene expression-inhibition compound (or at least onenucleic acid comprising a sequence encoding the miR gene product or miRgene expression-inhibition compound) (e.g., 0.1 to 90% by weight), or aphysiologically-acceptable salt thereof, mixed with apharmaceutically-acceptable carrier. In certain embodiments, thepharmaceutical composition of the invention additionally comprises oneor more anti-cancer agents (e.g., chemotherapeutic agents). Thepharmaceutical formulations of the invention can also comprise at leastone miR gene product or miR gene expression-inhibition compound (or atleast one nucleic acid comprising a sequence encoding the miR geneproduct or miR gene expression-inhibition compound), which areencapsulated by liposomes and a pharmaceutically-acceptable carrier.

Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude, e.g., physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (such as, for example,calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calciumor sodium salts (for example, calcium chloride, calcium ascorbate,calcium gluconate or calcium lactate). Pharmaceutical compositions ofthe invention can be packaged for use in liquid form, or can belyophilized.

For solid pharmaceutical compositions of the invention, conventionalnontoxic solid pharmaceutically-acceptable carriers can be used; forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administrationcan comprise any of the carriers and excipients listed above and 10-95%,preferably 25%-75%, of the at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisingsequences encoding them). A pharmaceutical composition for aerosol(inhalational) administration can comprise 0.01-20% by weight,preferably 1%-10% by weight, of the at least one miR gene product or miRgene expression-inhibition compound (or at least one nucleic acidcomprising a sequence encoding the miR gene product or miR geneexpression-inhibition compound) encapsulated in a liposome as describedabove, and a propellant. A carrier can also be included as desired;e.g., lecithin for intranasal delivery.

The pharmaceutical compositions of the invention can further compriseone or more anti-cancer agents. In a particular embodiment, thecompositions comprise at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisinga sequence encoding the miR gene product or miR geneexpression-inhibition compound) and at least one chemotherapeutic agent.Chemotherapeutic agents that are suitable for the methods of theinvention include, but are not limited to, DNA-alkylating agents,anti-tumor antibiotic agents, anti-metabolic agents, tubulin stabilizingagents, tubulin destabilizing agents, hormone antagonist agents,topoisomerase inhibitors, protein kinase inhibitors, HMG-CoA inhibitors,CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinaseinhibitors, antisense nucleic acids, triple-helix DNAs, nucleic acidsaptamers, and molecularly-modified viral, bacterial and exotoxic agents.Examples of suitable agents for the compositions of the presentinvention include, but are not limited to, cytidine arabinoside,methotrexate, vincristine, etoposide (VP-16), doxorubicin (adriamycin),cisplatin (CDDP), dexamethasone, arglabin, cyclophosphamide, sarcolysin,methylnitrosourea, fluorouracil, 5-fluorouracil (5FU), vinblastine,camptothecin, actinomycin-D, mitomycin C, hydrogen peroxide,oxaliplatin, irinotecan, topotecan, leucovorin, carmustine,streptozocin, CPT-11, taxol, tamoxifen, dacarbazine, rituximab,daunorubicin, 1-β-D-arabinofuranosylcytosine, imatinib, fludarabine,docetaxel and FOLFOX4.

In one embodiment, the method comprises providing a test agent to a celland measuring the level of at least one miR gene product associated withdecreased expression levels in cancerous cells. An increase in the levelof the miR gene product in the cell, relative to a suitable control(e.g., the level of the miR gene product in a control cell), isindicative of the test agent being an anti-cancer agent.

Suitable agents include, but are not limited to drugs (e.g , smallmolecules, peptides), and biological macromolecules (e.g., proteins,nucleic acids). The agent can be produced recombinantly, synthetically,or it may be isolated (i.e., purified) from a natural source. Variousmethods for providing such agents to a cell (e.g., transfection) arewell known in the art, and several of such methods are describedhereinabove. Methods for detecting the expression of at least one miRgene product (e.g., Northern blotting, in situ hybridization, RT-PCR,expression profiling) are also well known in the art. Several of thesemethods are also described herein.

EXAMPLES

The invention may be better understood by reference to the followingnon-limiting examples, which serve to illustrate but not to limit thepresent invention.

The data herein show that activated human mononuclear phagocytes andTHP-1 cells release microvesicles that induce the survival anddifferentiation of freshly isolate monocytes. While not wishing to bebound by theory, the inventors herein believe that under specificinflammatory diseases, the content of the microvesicles may be alteredto rapidly induce a response. The data also show that microvesiclescirculate in human peripheral blood. The circulating microvesiclesregulate normal cellular homeostasis, and circulate instructions todistant cells during tissue injury and inflammation.

The microvesicles may serve as biomarkers for disease etiology andsystemic mediators of the innate immune response. It is thus beneficialto be able to obtain similar information through the isolation ofmicrovesicles in the peripheral blood instead of obtaining tissuethrough invasive procedures. Also, understanding the normal signature ofmicrovesicles in the peripheral blood provides a basis for understandingevents during acute inflammatory events.

As shown herein, aberrant macrophage differentiation contributes todisruption in immune homeostasis. Since monocyte maturation is inducedby GM-CSF or M-CSF, the inventors initiated studies to understand themechanisms and differences between GM-CSF- and M-CSF-mediateddifferentiation. The commitment to differentiate in response to GM-CSFbut not M-CSF was rapid and irreversible (data not shown). ContinuousGM-CSF stimulation was not required for this effect as only 4 hours oftreatment induced macrophage differentiation. Similar observations wereobtained in PMA-treated THP1 cells used as a model of macrophagedifferentiation.

Thus, the inventors determined that at least one factor was secretedupon inducing differentiation that either maintained signals oractivated other cells to differentiate. Therefore, monocytes or THP1cells were exposed to GM-CSF for 4 h or PMA for 1 h, respectively, afterwhich cells were washed and placed in minimal media without stimulus.After 24 hours, the culture supernatants were collected and added toundifferentiated monocytes or THP1 cells. Notably, supernatants fromPMA-treated THP1 cells or GM-CSF-treated monocytes differentiatedmonocytes and THP1 cells (data not shown).

Using the Bioplex suspension array system to detect up to 27 differentcytokines in the culture supernatants, the inventors failed to detect aresponsible cytokine. Since the inventors differentiated the growthfactor-independent THP1 cell line with GM-CSF-stimulated monocytesupernatants, the inventors concluded that a cytokine/growth factor wasnot responsible for this effect. The inventors next investigated thepossibility that microvesicles were secreted in the culture supernatantto mediate myeloid maturation.

As shown in FIG. 1, monocytes treated with GM-CSF for 24 hours releasedsignificant numbers of microvesicles (dark dots) in the culturesupernatant compared to untreated monocytes (light dots).

Similarly, PMA-treated THP1 cells also secreted microvesicles duringdifferentiation (data not shown). In particular, FIG. 1 shows thedifferentiation induced release of microvesicles from macrophages.Peripheral blood monocytes (PBM) were untreated (light) or treated withGM-CSF (dark) for 24 h. Cell-free supernatant was collected andultracentrifuged. The vesicles were resuspended in PBS and analyzed forsize on a flow cytometry. Prior to analysis, FSS and SSC parameters wereadjusted using 2 μm standard beads (not shown). Shown is representativedata from three different donors.

Microvesicles from PMA-treated THP1 cells were purified and added toeither freshly isolated monocytes or undifferentiated THP1 cells. Themicrovesicles alone induced macrophage differentiation in both celltypes as indicated by morphology (see FIGS. 2A-2C) and expression ofsurface antigens (data not shown).

The content of these microvesicles has been analyzed. The inventorsdetected the presence of miRNAs in the microvesicles from PMA-treatedTHP1 cells (data not shown).

The inventors also evaluated circulating microvesicles and miRNA in theperipheral blood of normal volunteers. Based on size, the inventorsfound three subpopulations of microvesicles in the circulation (FIG.3A). Macrophage-derived microvesicles were detected using antibodiesthat detect mannose receptor (CD206) and MHC II (FIG. 3B). Approximately40% of the total microvesicles (gated region) in the plasma are derivedfrom macrophages based on expression of either CD206 or MHCII (FIG. 3C).

The inventors further determined whether miRNA are contained in theperipheral blood microvesicles. We detected expression of numerousmiRNAs. The highest detected miRNAs are shown in FIG. 8 showing TableIII (n=51).

Notably, miR-146 is undetectable in the peripheral blood whereas miR-155expression was 80-fold lower than the highest expressing miRNA. Sinceboth miR-146 and miR-155 were elevated in our IPF patient samples, butwere low to undetectable in peripheral blood from normal donors,examination of circulating miRNAs may serve as a biomarker of disease.

It is now shown herein that circulating microvesicles contain miRNAs andthat circulating microvesicles can provide an avenue for the miRNAs toelicit cell-to-cell communication. The microvesicles housing miRNA canalso provide insight into the genetic basis of disease and can serve aspredictive biomarkers.

Also, microvesicles released during macrophage differentiation canmediate maturation of immature cells. Microvesicles collected duringmacrophage maturation mediate the differentiation and survival of humanmonocytes and contain RNA. Both miRNA and processed mRNA are responsiblefor the maturation signals imparted on immature cells.

Example—Plasma

Microvesicles are isolated from the plasma of normal healthyindividuals. RNA is isolated from both the microvesicles and matchedmononuclear cells and profiled for 420 known mature miRNAs by real-timePCR. Hierarchal cluster analysis of the data sets indicated significantdifferences in miRNA expression between peripheral blood mononuclearcells (PBMC) and plasma microvesicles.

We observed 104 and 75 miRNAs significantly expressed in themicrovesicles and PBMC, respectively. Notably, 33 miRNAs werespecifically expressed microvesicles compared to the PBMC. The miRNAwere subjected to computational modeling to determine the biologicalpathways regulated by the detected miRNAs. The majority of the microRNAsexpressed in the microvesicles from the blood were predicted to regulatecellular differentiation of blood cells and metabolic pathways.Interestingly, a select few microRNAs are predicted to be importantmodulators of immune function.

This example is the first to identify and define miRNA expression incirculating plasma microvesicles of normal subjects.

Recent evidence reveals that genetic exchange of mRNA and miRNA betweencells can be accomplished through exosome-mediated transfer (PMID:17486113). Microvesicles are small exosomes/vesicles of endocytic originreleased by normal healthy or damaged cell types (PMID: 17337785, PMID:17409393, PMID: 16791265). Microvesicles are shed from the plasmamembrane into the extracellular environment to facilitate communicationbetween cells. Despite their small size (50nm to 1 μm) microvesicles areenriched in bioactive molecules and are suspected to contain nucleicacid and/or protein; these cell particles play a role in growth,differentiation and cancer progression (PMID: 16453000). In theperipheral blood, two-thirds of microvesicles are derived fromplatelets. Platelet-derived microvesicles play a role in angiogenesisand the metastatic spread of cancers such as lung cancer (PMID:15499615). Platelet-derived microvesicles induce an immune response uponregulating gene expression in hematopoietic, endothelial, and monocyticcells (PMID: 17378242, PMID: 17127485).

Interestingly, a connection between microvesicles and miRNA has beenrecently made. Recently, Valadi and colleagues reported that vesiclesreleased from human and murine mast cell lines contain over 1200 mRNAand approximately 121 miRNA molecules (PMID: 17486113) In contrast, thepresent invention relates to naturally occurring human plasma and bloodmicrovesicles containing microRNA that leads to biological effects exvivo.

FIG. 8—Table I shows that microRNAs that are important in humandiseases, including cancer and non-cancer applications. The microRNAmolecules associated with increase expression in disease tissue butnormally with low native or undetectable expression in human plasmamicrovesicles (Table I, shown in FIG. 6) provides the opportunity todefine changes in health and disease and may be effective biomarkers(Bold, Increase Expression Column). Similarly, normally abundantmicroRNAs may decrease in human plasma microvesicles to reflect thedecrease observed in tissue (Bold, Decrease Expression Column).

Considerable evidence demonstrates the importance of miRNA as aninevitable cornerstone of the human genetic system. Employing the use ofmicrovesicles to transfer genetic material would be an efficienttransfer method within the human body. Microvesicular transport ofmiRNAs would enable communication at long distance.

Methods

Blood collection and microvesicle isolation. Peripheral blood (40 cc)was collected in EDTA tubes from 24 females and 27 males healthynon-smoking Caucasian donors following informed consent. Collection ofthe blood occurred either between morning and early afternoon. Themedian age for female donors was 29 as well as for male donors. Theperipheral blood was diluted 1:1 with sterile low endotoxin PBS, layeredover ficoll-hypaque (d=1.077), and centrifuged as previously described(PMID: 16931806). The mononuclear cell fraction was washed once in PBS.The microvesicles were purified from the plasma. Briefly, the vesicleswere concentrated by centrifugation at 160,000×g for lhr at 4° C. (PMID:10648405).

RNA Extraction. Total RNA was isolated by Trizol (Invitrogen, Carlsbad,CA) extraction method. To increase the yield of small RNAs, the RNA wasprecipitated overnight. RNA concentration was determined and RNAintegrity was a determined by capillary electrophoresis on an Agilent2100 Bioanalyzer (Agilent Technologies, Inc, Santa Clara, Calif.). ForRNA isolated from mononuclear cells, only a RNA integrity number (RIN)≧9 was used. Since the intact 18s and 28s rRNA was variable in themicrovesicles, the RIN was not a constraint for these samples.

miRNA profiling by quantitative PCR. The expression of 420 mature humanmiRNAs was profiled by real-time PCR. RNA (50 ng) was converted to cDNAby priming with a mixture of looped primers to 420 known human maturemiRNAs (Mega Plex kit, Applied Biosystems, Foster City, Calif.) usingpreviously published reverse transcription conditions (PMID: 18158130).As there is no known control miRNA in microvesicles, several internalcontrols were examined. Primers to the internal controls, smallnucleolar (sno)RNA U38B, snoRNA U43, small nuclear (sn)RNA U6 as well as18S and 5S rRNA were included in the mix of primers.

The expression was profiled using an Applied Biosystems 7900HT real-timePCR instrument equipped with a 384 well reaction plate. Liquid-handlingrobots and the Zymak Twister robot were used to increase throughput andreduce error. Real-time PCR was performed using standard conditions.

Flow Cytometry. Peripheral blood microvesicles were directlyimmunostained from plasma without concentration by centrifugation. Todetermine the cellular origin, 0.5 cc plasma was immunostained per panelof antibodies. Panel I contained antibodies recognizing CD66b-FITC(neutrophil), CD202b (Tie2)-PE (endothelial), CD206 PE-Cy5(macrophage/dendritic), CD79a-APC (B-cell), and CD14 Pe-Cy7 (monocyte).Panel II contained antibodies to CD41a-PE-Cy5 (platelet), CCR2-APC(monocyte), CCR3-PE (dendritic cell), CCRS-PE-Cy7 (macrophage), andCD3-Alexa 610 (T-cell). Panel III contained isotype control antibodies.The samples were analyzed on BD Aria flow cytometer (BD Biosciences SanJose, Calif.). Data was expressed as percent of gated cells.

Statistical analysis. To reduce background noise, the miRNAs in which80% of individual observations had a raw CT score greater than 35 werenot considered during the data analysis. The internal controls (18S, 5S,snoRNA U38B, snoRNA U43, and snRNA U6) were highly variable in theplasma microvesicles as well as significantly different levels ofexpression in plasma microvesicles versus peripheral blood mononuclearcells (PBMC).

Thus, to reduce bias caused by using a certain miRNA as a normalizationcorrection factor and to reduce the sample variations among RT-PCRarrays, the miRNAs were compared between plasma microvesicles and PBMCbased on their relative expression to the overall miRNA expression oneach array using median normalization analysis (PMID: 16854228).Controlling gender and age of the donors, linear mixed models were usedto estimate the difference of specific miRNA between plasmamicrovesicles and PBMC. Fold-change was calculated based on theestimated mean difference.

Heat maps were generated using the miRNA that passed the filteringcriteria for each tissue and miRNAs were subjected to hierarchicalclustering based on their relative mean expression. miRNA expression wasalso ranked based on their raw CT score for plasma microvesicles andPBMC. Additional statistical analysis such as ANOVA was performed todetermine miRNAs that are significant expressed between the twotreatment groups

Pathway analysis and prediction. Predicted miRNAs targets weredetermined using miRanda (microrna.sanger.ac.uk/targets/v5/). Based onthe miRanda algorithm, a score is generated for each target, only scoresgreater than 17 were furthered analyzed using Ingenuity Pathway Analysissoftware (Ingenuity Systems, Redwood City, Calif.). Using this software,canonical pathways were determined based on targets of the miRNAs. Thedataset was examined to determine associated pathways based on geneontology of miRNA's targets.

Results

Peripheral Blood Microvesicle Subpopulations

Initially, we examined the cellular origin of microvesicles within theperipheral blood of normal healthy individuals. Using flow cytometry, wefound that the majority of the peripheral blood microvesicles areplatelet-derived (FIG. 4), as previously reported (PMID: 10648405).

We also observed a second large population of microvesicles that werederived from mononuclear cell phagocyte lineage. This population wasimmunostained with antibodies that detected surface antigens onmononuclear phagocytes. Notably, only a small percentage of theperipheral blood microvesicles were derived from T-cells andneutrophils. We failed to detect vesicles that originated from B-cells(data not shown). Of interest, we detected a small subpopulation ofmicrovesicles that expressed surface antigens from endothelial cells.

miRNA Expression in Plasma Microvesicles and PBMC

To test whether miRNAs are contained in the microvesicle compartmentwithin the peripheral blood to enable communication and influencegenetic changes between different tissues within the body, we performedmiRNA profiling on the purified microvesicles from the plasma. Weanalyzed all subpopulations of microvesicles from 51 non-smoking healthyindividuals comprising of 27 males and 24 females. In order to determinewhether there would be differences in miRNA expression betweenmicrovesicles and PBMC, we also purified the PBMC from each donor.Real-time PCR analysis was performed to examine the expression of 420miRNAs. The filtered data was subjected to hierarchal cluster analysiscomparing the miRNA expression profile between the PBMC and plasmasamples (FIG. 5A).

All but three PBMC samples clustered separately from the microvesiclesamples, indicating that the miRNA expression profile between the twogroups was significantly different. Based on filtering criteria toreduce background noise, we found 104 and 75 miRNAs expressed in themicrovesicles and PBMC samples, respectively (FIGS. 5B and 5C).

Of these miRNAs, 71 were shared among each sample group (FIG. 5D).Notably, only two miRNAs miR-031 and 29c were expressed solely in thePBMC samples whereas four miRNAs (miR -127, -134, -485-5p, and -432)were uniquely expressed in the plasma fraction. All 104 miRNAs that arenormally expressed in the plasma are shown (Table II, shown in FIG. 7).

Age and Gender Effects

We did not observe age and/or gender effects in miRNA expression fromeither sample group. Notably, the median age for both female and maledonors was 29 years. The oldest individual was 58 years old, while theyoungest was 21 years of age. Thus, we furthered stratified the data toexamine differences. Examination between age-matched samples did notreveal any significant effects on miRNA expression between PBMC andmicrovesicles samples. While controlling gender, we also compared theupper quartile of age with the lower quartile of age, mean age for eachgroup was 48.9±6.2 and 21.9±1.2, respectively. However, we failed todetect significant differences in miRNA expression between the samplessets based on age (data not shown).

Comparison of miRNA Expression in PBMC and Microvesicles

Shown in Table III, FIG. 8, is the top ten expressed miRNAs in theplasma microvesicles and the PBMC from all individuals. For plasma thetop ten expressed miRNAs in descending order are miR-223, -484, -191,-146a, -016, -026a, -222, -024, -126, and -32. Whereas, miR-223, -150,-146b, -016, -484, -146a, -191, -026a, -019b, and -020a were highlyexpressed in the PBMC. The top ten expressed miRNAs in the microvesicleswere detected in 100% of the individuals. However, in the PBMC samples,all but miR-150 (98% of donors) and miR-484 (89% of donors) wereobserved in 100% of the individuals.

We also found that six of these miRs (miR-223, miR-484, miR-191,miR-146a, miR-26a, and miR-16) are shared among the top ten in both PBMCand microvesicles. Notably, miR-223 is the most prominently expressedmiR in both compartments. Based on ranking analysis for each individualdonor to determine the frequency in which the specific miRNA appeared inthe top ten expressed miRNA, miR-223 had a frequency of 100% in bothPBMC and microvesicles. Despite expression of miR-486 being the in thetop ten expressed miRNAs in the plasma microvesicles, this miRNA wasfound to be expressed in the top ten of only 20% of the individualsprofiled. Interestingly, the highly expressed miRNAs in the plasmamicrovesicles were not identified as tissue-specific miRs.

We further examined the collective function of the miRs in microvesiclesand PBMC with a ranking score greater than arbitrary values of >66%and >88%, respectively (natural cut-offs from the data set). Based onthis criterion, we further examined the top 9 ranked miRs from themicrovesicles and PBMC samples. Thus, we analyzed the combined functionof miR-223, -484, -191, -146a, -016, -026a, -222, -024, and -126 foundin the plasma. For PBMC, we examined the combined function of thefollowing miRNAs, miR-223, -150, -146b, -016, -484, -146a, -191, -026a,and -019b. Using the Sanger miRBase Target version 5, we found 1578predicted targets of the combined miRs for the plasma microvesicles(data not shown). These combined targets were subjected to computationalanalysis to determine the pathways that they collectively regulate.Using the Ingenuity Pathway Analysis (IPA) software, we found canonicalpathways involved in metabolism and regulation of the acquired immunesystem were highly regulated by the expression of these shown in miRNAs(Table IV, shown in FIG. 9, top).

Of the nine miRNAs examined from the PBMC fraction, we found 1857predicted mRNA targets (data not shown). Ultimately the top fivecanonical pathways regulated by these miRNAs are various amino acid andlipid metabolic pathways, among others (Table IV, shown in FIG. 9, top).We also found common predicted targets from Sanger miRBase andTargetScan and determined their function (Table IV, shown in FIG. 9,bottom).

We next examined which miRNAs were differentially expressed betweenmicrovesicles and PBMC. We found 20 miRNAs had more than a three-foldincrease in expression in the PBMC fraction compared to themicrovesicles samples (Table V, shown in FIG. 10). In contrast, 15miRNAs were significantly expressed in the plasma microvesicles comparedto PBMC.

FIG. 11: Table VI shows the average normalized data for all miRNAs(detector name) expressed in the PBMC and the plasma with standarddeviation for each.

Discussion

In these examples, the inventors now show that miRs circulate inmicrovesicles under normal homeostatic conditions in the peripheralblood. Here, we demonstrate 104 miRs expressed in plasma microvesiclesand miR expression was significantly different from PBMC. To date,numerous studies demonstrate the ability of miRs to regulate manycellular functions. However, these studies largely imply that the miRstays within its host cell to elicit an effect (PMID: 17923084). Ourdata indicates that the miRNAs contained in the microvesicles may becommunication signals to distant cells to regulate cellular homeostasis.

These miRNAs in the microvesicles may circulate to different tissuetargets. Further examination of the highest expressed miRNAs in theplasma microvesicles, demonstrate that many of these function toregulate hematopoiesis and cellular differentiation programs (Table III,shown in FIG. 8). For instances, expression of miR-223 regulatesmyeloid, granulocytic and osteoclasts differentiation (PMID: 18278031,PMID: 17471500, PMID: 16325577). It also appears to have a role inhematopoietic stem cell proliferation (PMID: 18278031). Interestingly,miR-223 is loss in acute myelogenous leukemia (AML) (PMID: 18056805). Incontrast, downregulation of miR-126 occurs during megakaryocytedifferentiation (PMID: 16549775). Notably, expression of miR-24 isregulated by TGF-β which is a potent positive and negative regulator ofhematopoiesis (PMID: 16123808, PMID: 18353861). Both miR-24 and miR-16expressed in the microvesicles regulates red cell production (PMID:17906079, PMID: 17976518), while miR-16 also modulates lymphoiddevelopment (PMID: 16616063). Loss of miR-16 expression has beenextensively examined in chronic lymphocytic leukemia (CLL) (PMID:17327404, PMID: 17351108).

Many miRs expressed in the plasma microvesicles also regulate theprogression of the cell cycle proteins (PMID: 18365017 PMID: 17914108).MiR-222 targets p27Kipl (PMID: 17914108) while miR-24 suppresses p16(INK4a) (PMID: 18365017). Increased expression of miR-16 results in theaccumulation of cells in GO/G1 phase of the cell cycle (PMID: 16123808).In contrast, expression of miR-126 in breast cancer cells increasescellular proliferation and tumor growth but inhibits metastases (PMID:18185580). This occurs through the regulation of vascular cell adhesionmolecule-1 (VCAM1) (PMID: 18227515).

Unlike the other miRs highly expressed in the plasma microvesicles,miR-146a appears to function at a different level. While it has beensuggested that miR-146a acts as a tumor suppressor and loss of this miRis associated with the development of prostate cancer (PMID: 18174313),miR-146a also modulates immune function (PMID: 16885212, PMID:18057241). It is possible that expression of this miR in the plasmamicrovesicles defines immune regulatory function (Table IV, shown inFIG. 9).

Based on IPA analysis examining gene ontology of targets, the topassociated networks predicted to be influenced by miR-146a expression iscellular proliferation, immune and lymphatic system development andfunction. In addition, this miR is predicted to regulate innate immuneresponses. From the analysis, we found that LPS/IL-1 and toll-likereceptor signaling are among the top five canonical pathways predictedto be regulated by this mir-146a.

To date, there is no known function for miR-484 or miR-486. Similar tomiR-146a, miR-484 and miR-486 appear to function as a modulator ofimmune responsiveness. Notably, miR-484 is the second highest expressedmiR in the microvesicles fraction based on relative expression.Prediction modeling indicates that this miR has multiple functions. Likemany of the other miRs expressed in the microvesicles, miR-484 ispredicted to regulate hematopoiesis. In particular, NK cell signalingand IL-4 signaling pathways are predicted to be targets of miR-484,while miR-486 is proposed to regulate antigen presentation. In addition,miR-486 appears to regulate cell differentiation, proliferation andgrowth.

While we detected 104 miRs in the plasma microvesicles, there were manythat were undetectable from the total miRs profiled. Undetectable miRsin plasma microvesicles may also serve as disease biomarkers. Recently,Lawrie et al. reported that miRs were detected in the plasma of patientswith B-cell lymphoma (PMID: 18318758). This study, indicated thatmiR-155, miR-210 and miR-21 were elevated in the plasma from thesepatients and miR-21 correlated with relapse. Based on this study, wedetected miR-155 and miR-21 in normal individuals, but did not findmiR-210. Interestingly, we found that 75% of individuals expressedmiR-155 and 60% expressed miR-21 in the plasma (data not shown).

Thus, for these miRs to be used as predictive markers of disease, eachindividual would require a baseline prior to detection of disease. Thus,expression of miR-210 may serve as a better marker of B-cell lymphoma.Additional relationships may exist. For instance, miR-203 wasundetectable in plasma microvesicles. Elevated expression of this miR isassociated with bladder carcinoma and colon adenocarcinoma and may bethus used as a biomarker (PMID: 18230780, PMID: 17826655).

A converse relation may exist for plasma miRs that are normallyexpressed then lost with disease. For example, in acute lymphocyticleukemia (ALL), miR-223 is downregulated (PMID: 18056805). Since miR-223is the most prominent miR expressed in the plasma microvesicles, itsreduced expression may be useful as a diagnostic marker in ALL. Inaddition, miRs-15a/16 are lost or downregulated in chronic lymphocyticleukemia (CLL) (PMID: 18362358). While we found miR-16 was expressed inall healthy individuals that were examined, miR-15a was only expressedin 44% of the individuals profiled (data not shown).

It is of interest that we did not detect tissue specific miRNAs in theblood of normal individuals (PMID: 18025253). The majority of themicrovesicles from normal individuals are derived from blood cells. Wedid detect a small percentage of microvesicles derived from endothelialcells. The endothelial-derived microvesicles may increase uponendothelial cell damage. Likewise, the detection of tissue specific miRsand microvesicles in the peripheral blood may be a frequent event upontissue damage. Since tumors produce microvesicles (PMID: 16283305),these may be detected in the peripheral blood.

While it has been reported that miRs are detected in the plasma (PMID:18318758), this is the first study to characterize all known miRs fromthe plasma. In this study, we controlled race as a factor.

Testing the presence, absence or alterations in levels of miRs inperipheral fluids and/or blood can be useful as biomarkers to examinevarious diseases, to identify unique miRNA profiles, and to be apredictor of disease. The circulating miRs contained in themicrovesicles have a vital function in regulating homeostasis productionof blood cells as well as metabolic functions

The relevant teachings of all publications cited herein that have notexplicitly been incorporated by reference, are incorporated herein byreference in their entirety. While this invention has been particularlyshown and described with references to preferred embodiments thereof, itwill be understood by those skilled in the art that various changes inform and details may be made therein without departing from the scope ofthe invention encompassed by the appended claims.

While the invention has been described with reference to various andpreferred embodiments, it should be understood by those skilled in theart that various changes may be made and equivalents may be substitutedfor elements thereof without departing from the essential scope of theinvention. In addition, many modifications may be made to adapt aparticular situation or material to the teachings of the inventionwithout departing from the essential scope thereof. Therefore, it isintended that the invention not be limited to the particular embodimentdisclosed herein contemplated for carrying out this invention, but thatthe invention will include all embodiments falling within the scope ofthe claims.

The miRs of interest are listed in public databases. In certainpreferred embodiments, the public database can be a central repositoryprovided by the Sanger Institutewww.http://microrna.sanger.ac.uk/sequences/to which miR sequences aresubmitted for naming and nomenclature assignment, as well as placementof the sequences in a database for archiving and for online retrievalvia the world wide web. Generally, the data collected on the sequencesof miRs by the Sanger Institute include species, source, correspondinggenomic sequences and genomic location (chromosomal coordinates), aswell as full length transcription products and sequences for the maturefully processed miRNA (miRNA with a 5′ terminal phosphate group).Another database can be the GenBank database accessed through theNational Center for Biotechnology Information (NCBI) website, maintainedby the National Institutes of Health and the National Library ofMedicine. These databases are fully incorporated herein by reference.

microR *Biogenesis miRBase byproducts that Mature SEQ are at low level,Sequence ID function unknown Accession # Mature Sequence NO hsa-let-7a*MIMAT0004481 CUAUACAAUCUACUGUCUUUC 1 hsa-let-7a-1 MIMAT0000062UGAGGUAGUAGGUUGUAUAGUU 2 hsa-let-7a-2 MIMAT0000062UGAGGUAGUAGGUUGUAUAGUU 3 hsa-let-7a-3 MIMAT0000062UGAGGUAGUAGGUUGUAUAGUU 4 hsa-let-7b MIMAT0000063 UGAGGUAGUAGGUUGUGUGGUU5 hsa-let-7b* MIMAT0004482 CUAUACAACCUACUGCCUUCCC 6 hsa-let-7cMIMAT0000064 UGAGGUAGUAGGUUGUAUGGUU 7 hsa-let-7c* MIMAT0004483UAGAGUUACACCCUGGGAGUUA 8 hsa-let-7d MIMAT0000065 AGAGGUAGUAGGUUGCAUAGUU9 hsa-let-7d* MIMAT0004484 CUAUACGACCUGCUGCCUUUCU 10 hsa-let-7eMIMAT0000066 UGAGGUAGGAGGUUGUAUAGUU 11 hsa-let-7e* MIMAT0004485CUAUACGGCCUCCUAGCUUUCC 12 hsa-let-7f-1 MIMAT0000067UGAGGUAGUAGAUUGUAUAGUU 13 hsa-let-7f-1* MIMAT0004486CUAUACAAUCUAUUGCCUUCCC 14 hsa-let-7f-2 MIMAT0000067UGAGGUAGUAGAUUGUAUAGUU 15 hsa-let-7f-2* MIMAT0004487CUAUACAGUCUACUGUCUUUCC 16 hsa-let-7g MIMAT0000414 UGAGGUAGUAGUUUGUACAGUU17 hsa-let-7g* MIMAT0004584 CUGUACAGGCCACUGCCUUGC 18 hsa-let-7iMIMAT0000415 UGAGGUAGUAGUUUGUGCUGUU 19 hsa-let-7i* MIMAT0004585CUGCGCAAGCUACUGCCUUGCU 20 hsa-mir-009-1 MIMAT0000441UCUUUGGUUAUCUAGCUGUAUGA 21 hsa-mir-009-1* MIMAT0000442AUAAAGCUAGAUAACCGAAAGU 22 hsa-mir-009-2 MIMAT0000441UCUUUGGUUAUCUAGCUGUAUGA 23 hsa-mir-009-3 MIMAT0000441UCUUUGGUUAUCUAGCUGUAUGA 24 hsa-mir-010a MIMAT0000253UACCCUGUAGAUCCGAAUUUGUG 25 hsa-mir-010a* MIMAT0004555CAAAUUCGUAUCUAGGGGAAUA 26 hsa-mir-015a MIMAT0000068UAGCAGCACAUAAUGGUUUGUG 27 hsa-mir-015b MIMAT0000417UAGCAGCACAUCAUGGUUUACA 28 hsa-mir-015b* MIMAT0004586CGAAUCAUUAUUUGCUGCUCUA 29 hsa-mir-016-1 MIMAT0000069UAGCAGCACGUAAAUAUUGGCG 30 hsa-mir-016-1* MIMAT0004489CCAGUAUUAACUGUGCUGCUGA 31 hsa-mir-016-2 MIMAT0000069UAGCAGCACGUAAAUAUUGGCG 32 hsa-mir-016-2* MIMAT0004518CCAAUAUUACUGUGCUGCUUUA 33 hsa-mir-017-3-p MIMAT0000071ACUGCAGUGAAGGCACUUGUAG 34 hsa-mir-017-5-p MIMAT0000070CAAAGUGCUUACAGUGCAGGUAG 35 hsa-mir-018a MIMAT0000072UAAGGUGCAUCUAGUGCAGAUAG 36 hsa-mir-018a* MIMAT0002891ACUGCCCUAAGUGCUCCUUCUGG 37 hsa-mir-019a MIMAT0000073UGUGCAAAUCUAUGCAAAACUGA 38 hsa-mir-019b-1 MIMAT0000074UGUGCAAAUCCAUGCAAAACUGA 39 hsa-mir-019b-1* MIMAT0004491AGUUUUGCAGGUUUGCAUCCAGC 40 hsa-mir-019b-2 MIMAT0000074UGUGCAAAUCCAUGCAAAACUGA 41 hsa-mir-019b-2* MIMAT0004492AGUUUUGCAGGUUUGCAUUUCA 42 hsa-mir-020a MIMAT0000075UAAAGUGCUUAUAGUGCAGGUAG 43 hsa-mir-020a* MIMAT0004493ACUGCAUUAUGAGCACUUAAAG 44 hsa-mir-020b MIMAT0001413CAAAGUGCUCAUAGUGCAGGUAG 45 hsa-mir-021 MIMAT0000076UAGCUUAUCAGACUGAUGUUGA 46 hsa-mir-021* MIMAT0004494CAACACCAGUCGAUGGGCUGU 47 hsa-mir-023 a MIMAT0000078AUCACAUUGCCAGGGAUUUCC 48 hsa-mir-023 a* MIMAT0004496GGGGUUCCUGGGGAUGGGAUUU 49 hsa-mir-023b MIMAT0004587UGGGUUCCUGGCAUGCUGAUUU 50 hsa-mir-024-1 MIMAT0000080UGGCUCAGUUCAGCAGGAACAG 51 hsa-mir-024-1* MIMAT0000079UGCCUACUGAGCUGAUAUCAGU 52 hsa-mir-024-2 MIMAT0000080UGGCUCAGUUCAGCAGGAACAG 53 hsa-mir-024-2* MIMAT0004497UGCCUACUGAGCUGAAACACAG 54 hsa-mir-025 MIMAT0000081CAUUGCACUUGUCUCGGUCUGA 55 hsa-mir-025* MIMAT0004498AGGCGGAGACUUGGGCAAUUG 56 hsa-mir-026a-1 MIMAT0000082UUCAAGUAAUCCAGGAUAGGCU 57 hsa-mir-026a-1* MIMAT0004499CCUAUUCUUGGUUACUUGCACG 58 hsa-mir-026a-2 MIMAT0000082UUCAAGUAAUCCAGGAUAGGCU 59 hsa-mir-026a-2* MIMAT0004681CCUAUUCUUGAUUACUUGUUUC 60 hsa-mir-026b MIMAT0000083UUCAAGUAAUUCAGGAUAGGU 61 hsa-mir-026b* MIMAT0004500CCUGUUCUCCAUUACUUGGCUC 62 hsa-mir-027a MIMAT0000084UUCACAGUGGCUAAGUUCCGC 63 hsa-mir-027a* MIMAT0004501AGGGCUUAGCUGCUUGUGAGCA 64 hsa-mir-027b MIMAT0000419UUCACAGUGGCUAAGUUCUGC 65 hsa-mir-027b* MIMAT0004588AGAGCUUAGCUGAUUGGUGAAC 66 hsa-mir-028-3p MIMAT0004502CACUAGAUUGUGAGCUCCUGGA 67 hsa-mir-028-5p MIMAT0000085AAGGAGCUCACAGUCUAUUGAG 68 hsa-mir-029a MIMAT0000086UAGCACCAUCUGAAAUCGGUUA 69 hsa-mir-029a* MIMAT0004503ACUGAUUUCUUUUGGUGUUCAG 70 hsa-mir-029b-1 MIMAT0000100UAGCACCAUUUGAAAUCAGUGUU 71 hsa-mir-029b-1* MIMAT0004514GCUGGUUUCAUAUGGUGGUUUAGA 72 hsa-mir-029b-2 MIMAT0000100UAGCACCAUUUGAAAUCAGUGUU 73 hsa-mir-029b-2* MIMAT0004515CUGGUUUCACAUGGUGGCUUAG 74 hsa-mir-029b-3 MIMAT0000100UAGCACCAUUUGAAAUCAGUGUU 75 hsa-mir-029c MIMAT0000681UAGCACCAUUUGAAAUCGGUUA 76 hsa-mir-030a MIMAT0000087UGUAAACAUCCUCGACUGGAAG 77 hsa-mir-030a* MIMAT0000088CUUUCAGUCGGAUGUUUGCAGC 78 hsa-mir-030b MIMAT0000420UGUAAACAUCCUACACUCAGCU 79 hsa-mir-030b* MIMAT0004589CUGGGAGGUGGAUGUUUACUUC 80 hsa-mir-030c-1 MIMAT0000244UGUAAACAUCCUACACUCUCAGC 81 hsa-mir-030c-2 MIMAT0000244UGUAAACAUCCUACACUCUCAGC 82 hsa-mir-030c-2* MIMAT0004550CUGGGAGAAGGCUGUUUACUCU 83 hsa-mir-030d MIMAT0000245UGUAAACAUCCCCGACUGGAAG 84 hsa-mir-030d* MIMAT0004551CUUUCAGUCAGAUGUUUGCUGC 85 hsa-mir-031 MIMAT0000089 AGGCAAGAUGCUGGCAUAGCU86 hsa-mir-031* MIMAT0004504 UGCUAUGCCAACAUAUUGCCAU 87 hsa-mir-032MIMAT0000090 UAUUGCACAUUACUAAGUUGCA 88 hsa-mir-032* MIMAT0004505CAAUUUAGUGUGUGUGAUAUUU 89 hsa-mir-034a MIMAT0000255UGGCAGUGUCUUAGCUGGUUGU 90 hsa-mir-034a* MIMAT0004557CAAUCAGCAAGUAUACUGCCCU 91 hsa-mir-092a-1 MIMAT0000092UAUUGCACUUGUCCCGGCCUGU 92 hsa-mir-092a-1* MIMAT0004507AGGUUGGGAUCGGUUGCAAUGCU 93 hsa-mir-093 MIMAT0000093CAAAGUGCUGUUCGUGCAGGUAG 94 hsa-mir-093* MIMAT0004509ACUGCUGAGCUAGCACUUCCCG 95 hsa-mir-095 MIMAT0000094UUCAACGGGUAUUUAUUGAGCA 96 hsa-mir-096 MIMAT0000095UUUGGCACUAGCACAUUUUUGCU 97 hsa-mir-096* MIMAT0004510AAUCAUGUGCAGUGCCAAUAUG 98 hsa-mir-098 MIMAT0000096UGAGGUAGUAAGUUGUAUUGUU 99 hsa-mir-099b MIMAT0000689CACCCGUAGAACCGACCUUGCG 100 hsa-mir-099b* MIMAT0004678CAAGCUCGUGUCUGUGGGUCCG 101 hsa-mir-100 MIMAT0000098AACCCGUAGAUCCGAACUUGUG 102 hsa-mir-100* MIMAT0004512CAAGCUUGUAUCUAUAGGUAUG 103 hsa-mir-103-1 MIMAT0000101AGCAGCAUUGUACAGGGCUAUGA 104 hsa-mir-103-2 MIMAT0000101AGCAGCAUUGUACAGGGCUAUGA 105 hsa-mir-105-1 MIMAT0000102UCAAAUGCUCAGACUCCUGUGGU 106 hsa-mir-105-1* MIMAT0004516ACGGAUGUUUGAGCAUGUGCUA 107 hsa-mir-105-2 MIMAT0000102UCAAAUGCUCAGACUCCUGUGGU 108 hsa-mir-105-2* MIMAT0004516ACGGAUGUUUGAGCAUGUGCUA 109 hsa-mir-106a MIMAT0000103AAAAGUGCUUACAGUGCAGGUAG 110 hsa-mir-106a* MIMAT0004517CUGCAAUGUAAGCACUUCUUAC 111 hsa-mir-106b MIMAT0000680UAAAGUGCUGACAGUGCAGAU 112 hsa-mir-106b* MIMAT0004672CCGCACUGUGGGUACUUGCUGC 113 hsa-mir-107 MIMAT0000104AGCAGCAUUGUACAGGGCUAUCA 114 hsa-mir-122 MIMAT0000421UGGAGUGUGACAAUGGUGUUUG 115 hsa-mir-122* MIMAT0004590AACGCCAUUAUCACACUAAAUA 116 hsa-mir-125a-3p MIMAT0004602ACAGGUGAGGUUCUUGGGAGCC 117 hsa-mir-125a-5p MIMAT0000443UCCCUGAGACCCUUUAACCUGUGA 118 hsa-mir-125b-1 MIMAT0000423UCCCUGAGACCCUAACUUGUGA 119 hsa-mir-125b-1* MIMAT0004592ACGGGUUAGGCUCUUGGGAGCU 120 hsa-mir-125b-2 MIMAT0000423UCCCUGAGACCCUAACUUGUGA 121 hsa-mir-125b-2* MIMAT0004603UCACAAGUCAGGCUCUUGGGAC 122 hsa-mir-126 MIMAT0000445UCGUACCGUGAGUAAUAAUGCG 123 hsa-mir-126* MIMAT0000444CAUUAUUACUUUUGGUACGCG 124 hsa-mir-127-3p MIMAT0000446UCGGAUCCGUCUGAGCUUGGCU 125 hsa-mir-127-5p MIMAT0004604CUGAAGCUCAGAGGGCUCUGAU 126 hsa-mir-128-1 MIMAT0000424UCACAGUGAACCGGUCUCUUU 127 hsa-mir-128-2 MIMAT0000424UCACAGUGAACCGGUCUCUUU 128 hsa-mir-130a MIMAT0000425CAGUGCAAUGUUAAAAGGGCAU 129 hsa-mir-130a* MIMAT0004593UUCACAUUGUGCUACUGUCUGC 130 hsa-mir-130b MIMAT0000691CAGUGCAAUGAUGAAAGGGCAU 131 hsa-mir-130b* MIMAT0004680ACUCUUUCCCUGUUGCACUAC 132 hsa-mir-132 MIMAT0000426UAACAGUCUACAGCCAUGGUCG 133 hsa-mir-132* MIMAT0004594ACCGUGGCUUUCGAUUGUUACU 134 hsa-mir-133a-1 MIMAT0000427UUUGGUCCCCUUCAACCAGCUG 135 hsa-mir-133a-2 MIMAT0000427UUUGGUCCCCUUCAACCAGCUG 136 hsa-mir-133b MIMAT0000770UUUGGUCCCCUUCAACCAGCUA 137 hsa-mir-134 MIMAT0000447UGUGACUGGUUGACCAGAGGGG 138 hsa-mir-135b MIMAT0000758UAUGGCUUUUCAUUCCUAUGUGA 139 hsa-mir-135b* MIMAT0004698AUGUAGGGCUAAAAGCCAUGGG 140 hsa-mir-140-3p MIMAT0004597UACCACAGGGUAGAACCACGG 141 hsa-mir-140-5p MIMAT0000431CAGUGGUUUUACCCUAUGGUAG 142 hsa-mir-142-3p MIMAT0000434UGUAGUGUUUCCUACUUUAUGGA 143 hsa-mir-142-5p MIMAT0000433CAUAAAGUAGAAAGCACUACU 144 hsa-mir-143 MIMAT0000435 UGAGAUGAAGCACUGUAGCUC145 hsa-mir-143* MIMAT0004599 GGUGCAGUGCUGCAUCUCUGGU 146 hsa-mir-145MIMAT0000437 GUCCAGUUUUCCCAGGAAUCCCU 147 hsa-mir-145* MIMAT0004601GGAUUCCUGGAAAUACUGUUCU 148 hsa-mir-146a MIMAT0000449UGAGAACUGAAUUCCAUGGGUU 149 hsa-mir-146a* MIMAT0004608CCUCUGAAAUUCAGUUCUUCAG 150 hsa-mir-146b-3p MIMAT0004766UGCCCUGUGGACUCAGUUCUGG 151 hsa-mir-146b-5p MIMAT0002809UGAGAACUGAAUUCCAUAGGCU 152 hsa-mir-147 MIMAT0000251 GUGUGUGGAAAUGCUUCUGC153 hsa-mir-148a MIMAT0000243 UCAGUGCACUACAGAACUUUGU 154 hsa-mir-148a*MIMAT0004549 AAAGUUCUGAGACACUCCGACU 155 hsa-mir-148b MIMAT0000759UCAGUGCAUCACAGAACUUUGU 156 hsa-mir-148b* MIMAT0004699AAGUUCUGUUAUACACUCAGGC 157 hsa-mir-149 MIMAT0000450UCUGGCUCCGUGUCUUCACUCCC 158 hsa-mir-149* MIMAT0004609AGGGAGGGACGGGGGCUGUGC 159 hsa-mir-150 MIMAT0000451UCUCCCAACCCUUGUACCAGUG 160 hsa-mir-150* MIMAT0004610CUGGUACAGGCCUGGGGGACAG 161 hsa-mir-151-3p MIMAT0000757CUAGACUGAAGCUCCUUGAGG 162 hsa-mir-151-5p MIMAT0004697UCGAGGAGCUCACAGUCUAGU 163 hsa-mir-155 MIMAT0000646UUAAUGCUAAUCGUGAUAGGGGU 164 hsa-mir-155* MIMAT0004658CUCCUACAUAUUAGCAUUAACA 165 hsa-mir-181a-1 MIMAT0000256AACAUUCAACGCUGUCGGUGAGU 166 hsa-mir-181a-1* MIMAT0000270ACCAUCGACCGUUGAUUGUACC 167 hsa-mir-181a-2 MIMAT0000256AACAUUCAACGCUGUCGGUGAGU 168 hsa-mir-181a-2* MIMAT0004558ACCACUGACCGUUGACUGUACC 169 hsa-mir-181b-1 MIMAT0000257AACAUUCAUUGCUGUCGGUGGGU 170 hsa-mir-181b-2 MIMAT0000257AACAUUCAUUGCUGUCGGUGGGU 171 hsa-mir-181d MIMAT0002821AACAUUCAUUGUUGUCGGUGGGU 172 hsa-mir-182 MIMAT0000259UUUGGCAAUGGUAGAACUCACACU 173 hsa-mir-182* MIMAT0000260UGGUUCUAGACUUGCCAACUA 174 hsa-mir-183 MIMAT0000261UAUGGCACUGGUAGAAUUCACU 175 hsa-mir-183* MIMAT0004560GUGAAUUACCGAAGGGCCAUAA 176 hsa-mir-185 MIMAT0000455UGGAGAGAAAGGCAGUUCCUGA 177 hsa-mir-185* MIMAT0004611AGGGGCUGGCUUUCCUCUGGUC 178 hsa-mir-186 MIMAT0000456CAAAGAAUUCUCCUUUUGGGCU 179 hsa-mir-186* MIMAT0004612GCCCAAAGGUGAAUUUUUUGGG 180 hsa-mir-190 MIMAT0000458UGAUAUGUUUGAUAUAUUAGGU 181 hsa-mir-191 MIMAT0000440CAACGGAAUCCCAAAAGCAGCUG 182 hsa-mir-191* MIMAT0001618GCUGCGCUUGGAUUUCGUCCCC 183 hsa-mir-192 MIMAT0000222CUGACCUAUGAAUUGACAGCC 184 hsa-mir-192* MIMAT0004543CUGCCAAUUCCAUAGGUCACAG 185 hsa-mir-193a-3p MIMAT0000459AACUGGCCUACAAAGUCCCAGU 186 hsa-mir-193a-5p MIMAT0004614UGGGUCUUUGCGGGCGAGAUGA 187 hsa-mir-193b MIMAT0002819AACUGGCCCUCAAAGUCCCGCU 188 hsa-mir-193b* MIMAT0004767CGGGGUUUUGAGGGCGAGAUGA 189 hsa-mir-195 MIMAT0000461UAGCAGCACAGAAAUAUUGGC 190 hsa-mir-195* MIMAT0004615CCAAUAUUGGCUGUGCUGCUCC 191 hsa-mir-196a* MIMAT0004562CGGCAACAAGAAACUGCCUGAG 192 hsa-mir-196a-1 MIMAT0000226UAGGUAGUUUCAUGUUGUUGGG 193 hsa-mir-196a-2 MIMAT0000226UAGGUAGUUUCAUGUUGUUGGG 194 hsa-mir-196b MIMAT0001080UAGGUAGUUUCCUGUUGUUGGG 195 hsa-mir-197 MIMAT0000227UUCACCACCUUCUCCACCCAGC 196 hsa-mir-198 MIMAT0000228GGUCCAGAGGGGAGAUAGGUUC 197 hsa-mir-199a-3p MIMAT0000232ACAGUAGUCUGCACAUUGGUUA 198 hsa-mir-199a-5p MIMAT0000231CCCAGUGUUCAGACUACCUGUUC 199 hsa-mir-199a-5p MIMAT0000231CCCAGUGUUCAGACUACCUGUUC 200 hsa-mir-199b-3p MIMAT0004563ACAGUAGUCUGCACAUUGGUUA 201 hsa-mir-199b-5p MIMAT0000263CCCAGUGUUUAGACUAUCUGUUC 202 hsa-mir-200a MIMAT0000682UAACACUGUCUGGUAACGAUGU 203 hsa-mir-200a* MIMAT0001620CAUCUUACCGGACAGUGCUGGA 204 hsa-mir-200b MIMAT0000318UAAUACUGCCUGGUAAUGAUGA 205 hsa-mir-200b* MIMAT0004571CAUCUUACUGGGCAGCAUUGGA 206 hsa-mir-200c MIMAT0000617UAAUACUGCCGGGUAAUGAUGGA 207 hsa-mir-200c* MIMAT0004657CGUCUUACCCAGCAGUGUUUGG 208 hsa-mir-203 MIMAT0000264GUGAAAUGUUUAGGACCACUAG 209 hsa-mir-204 MIMAT0000265UUCCCUUUGUCAUCCUAUGCCU 210 hsa-mir-205 MIMAT0000266UCCUUCAUUCCACCGGAGUCUG 211 hsa-mir-210 MIMAT0000267CUGUGCGUGUGACAGCGGCUGA 212 hsa-mir-213 MIMAT0000256AACAUUCAACGCUGUCGGUGAGU 213 hsa-mir-214 MIMAT0000271ACAGCAGGCACAGACAGGCAGU 214 hsa-mir-214* MIMAT0004564UGCCUGUCUACACUUGCUGUGC 215 hsa-mir-216a MIMAT0000273UAAUCUCAGCUGGCAACUGUGA 216 hsa-mir-216b MIMAT0004959AAAUCUCUGCAGGCAAAUGUGA 217 hsa-mir-217 MIMAT0000274UACUGCAUCAGGAACUGAUUGGA 218 hsa-mir-218-1 MIMAT0000275UUGUGCUUGAUCUAACCAUGU 219 hsa-mir-218-1* MIMAT0004565AUGGUUCCGUCAAGCACCAUGG 220 hsa-mir-218-2 MIMAT0000275UUGUGCUUGAUCUAACCAUGU 221 hsa-mir-218-2* MIMAT0004566CAUGGUUCUGUCAAGCACCGCG 222 hsa-mir-221 MIMAT0000278AGCUACAUUGUCUGCUGGGUUUC 223 hsa-mir-221* MIMAT0004568ACCUGGCAUACAAUGUAGAUUU 224 hsa-mir-222 MIMAT0000279AGCUACAUCUGGCUACUGGGU 225 hsa-mir-222* MIMAT0004569CUCAGUAGCCAGUGUAGAUCCU 226 hsa-mir-223 MIMAT0000280UGUCAGUUUGUCAAAUACCCCA 227 hsa-mir-223* MIMAT0004570CGUGUAUUUGACAAGCUGAGUU 228 hsa-mir-224 MIMAT0000281CAAGUCACUAGUGGUUCCGUU 229 hsa-mir-302a MIMAT0000684UAAGUGCUUCCAUGUUUUGGUGA 230 hsa-mir-302a* MIMAT0000683ACUUAAACGUGGAUGUACUUGCU 231 hsa-mir-302b MIMAT0000715UAAGUGCUUCCAUGUUUUAGUAG 232 hsa-mir-302b* MIMAT0000714ACUUUAACAUGGAAGUGCUUUC 233 hsa-mir-302c MIMAT0000717UAAGUGCUUCCAUGUUUCAGUGG 234 hsa-mir-302c* MIMAT0000716UUUAACAUGGGGGUACCUGCUG 235 hsa-mir-302d MIMAT0000718UAAGUGCUUCCAUGUUUGAGUGU 236 hsa-mir-302d* MIMAT0004685ACUUUAACAUGGAGGCACUUGC 237 hsa-mir-302e MIMAT0005931 UAAGUGCUUCCAUGCUU238 hsa-mir-302f MIMAT0005932 UAAUUGCUUCCAUGUUU 239 hsa-mir-320aMIMAT0000510 AAAAGCUGGGUUGAGAGGGCGA 240 hsa-mir-320b-1 MIMAT0005792AAAAGCUGGGUUGAGAGGGCAA 241 hsa-mir-320b-2 MIMAT0005792AAAAGCUGGGUUGAGAGGGCAA 242 hsa-mir-320c-1 MIMAT0005793AAAAGCUGGGUUGAGAGGGU 243 hsa-mir-320c-2 MIMAT0005793AAAAGCUGGGUUGAGAGGGU 244 hsa-mir-320d-1 MIMAT0006764 AAAAGCUGGGUUGAGAGGA245 hsa-mir-320d-2 MIMAT0006764 AAAAGCUGGGUUGAGAGGA 246 hsa-mir-324-3pMIMAT0000762 ACUGCCCCAGGUGCUGCUGG 247 hsa-mir-324-5p MIMAT0000761CGCAUCCCCUAGGGCAUUGGUGU 248 hsa-mir-326 MIMAT0000756CCUCUGGGCCCUUCCUCCAG 249 hsa-mir-328 MIMAT0000752 CUGGCCCUCUCUGCCCUUCCGU250 hsa-mir-330-3p MIMAT0000751 GCAAAGCACACGGCCUGCAGAGA 251hsa-mir-330-5p MIMAT0004693 UCUCUGGGCCUGUGUCUUAGGC 252 hsa-mir-331-3pMIMAT0000760 GCCCCUGGGCCUAUCCUAGAA 253 hsa-mir-331-5p MIMAT0004700CUAGGUAUGGUCCCAGGGAUCC 254 hsa-mir-335 MIMAT0000765UCAAGAGCAAUAACGAAAAAUGU 255 hsa-mir-335* MIMAT0004703UUUUUCAUUAUUGCUCCUGACC 256 hsa-mir-339-3p MIMAT0004702UGAGCGCCUCGACGACAGAGCCG 257 hsa-mir-339-5p MIMAT0000764UCCCUGUCCUCCAGGAGCUCACG 258 hsa-mir-340 MIMAT0004692UUAUAAAGCAAUGAGACUGAUU 259 hsa-mir-340* MIMAT0000750UCCGUCUCAGUUACUUUAUAGC 260 hsa-mir-342-3p MIMAT0000753UCUCACACAGAAAUCGCACCCGU 261 hsa-mir-342-5p MIMAT0004694AGGGGUGCUAUCUGUGAUUGA 262 hsa-mir-345 MIMAT0000772GCUGACUCCUAGUCCAGGGCUC 263 hsa-mir-361-3p MIMAT0004682UCCCCCAGGUGUGAUUCUGAUUU 264 hsa-mir-361-5p MIMAT0000703UUAUCAGAAUCUCCAGGGGUAC 265 hsa-mir-370 MIMAT0000722GCCUGCUGGGGUGGAACCUGGU 266 hsa-mir-374a MIMAT0000727UUAUAAUACAACCUGAUAAGUG 267 hsa-mir-374b MIMAT0004955AUAUAAUACAACCUGCUAAGUG 268 hsa-mir-376a* MIMAT0003386GUAGAUUCUCCUUCUAUGAGUA 269 hsa-mir-376a-1 MIMAT0000729AUCAUAGAGGAAAAUCCACGU 270 hsa-mir-376a-2 MIMAT0000729AUCAUAGAGGAAAAUCCACGU 271 hsa-mir-376b MIMAT0002172AUCAUAGAGGAAAAUCCAUGUU 272 hsa-mir-376c MIMAT0000720AACAUAGAGGAAAUUCCACGU 273 hsa-mir-378 MIMAT0000732 ACUGGACUUGGAGUCAGAAGG274 hsa-mir-378* MIMAT0000731 CUCCUGACUCCAGGUCCUGUGU 275 hsa-mir-382MIMAT0000737 GAAGUUGUUCGUGGUGGAUUCG 276 hsa-mir-411 MIMAT0003329UAGUAGACCGUAUAGCGUACG 277 hsa-mir-411* MIMAT0004813UAUGUAACACGGUCCACUAACC 278 hsa-mir-423 MIMAT0004748UGAGGGGCAGAGAGCGAGACUUU 279 hsa-mir-423* MIMAT0001340AGCUCGGUCUGAGGCCCCUCAGU 280 hsa-mir-425-3p MIMAT0001343AUCGGGAAUGUCGUGUCCGCCC 281 hsa-mir-425-5p MIMAT0003393AAUGACACGAUCACUCCCGUUGA 282 hsa-mir-432 MIMAT0002814UCUUGGAGUAGGUCAUUGGGUGG 283 hsa-mir-432* MIMAT0002815CUGGAUGGCUCCUCCAUGUCU 284 hsa-mir-433 MIMAT0001627AUCAUGAUGGGCUCCUCGGUGU 285 hsa-mir-484 MIMAT0002174UCAGGCUCAGUCCCCUCCCGAU 286 hsa-mir-485-3p MIMAT0002176GUCAUACACGGCUCUCCUCUCU 287 hsa-mir-485-5p MIMAT0002175AGAGGCUGGCCGUGAUGAAUUC 288 hsa-mir-486-3p MIMAT0004762CGGGGCAGCUCAGUACAGGAU 289 hsa-mir-486-5p MIMAT0002177UCCUGUACUGAGCUGCCCCGAG 290 hsa-mir-487a MIMAT0002178AAUCAUACAGGGACAUCCAGUU 291 hsa-mir-487b MIMAT0003180AAUCGUACAGGGUCAUCCACUU 292 hsa-mir-532 MIMAT0002888CAUGCCUUGAGUGUAGGACCGU 293 hsa-mir-532-5p MIMAT0004780CCUCCCACACCCAAGGCUUGCA 294 hsa-mir-539 MIMAT0003163GGAGAAAUUAUCCUUGGUGUGU 295 hsa-mir-574-3p MIMAT0003239CACGCUCAUGCACACACCCACA 296 hsa-mir-574-5p MIMAT0004795UGAGUGUGUGUGUGUGAGUGUGU 297 hsa-mir-584 MIMAT0003249UUAUGGUUUGCCUGGGACUGAG 298 hsa-mir-628-3p MIMAT0003297UCUAGUAAGAGUGGCAGUCGA 299 hsa-mir-628-5p MIMAT0004809AUGCUGACAUAUUUACUAGAGG 300 hsa-mir-643 MIMAT0003313ACUUGUAUGCUAGCUCAGGUAG 301 hsa-mir-660 MIMAT0003338UACCCAUUGCAUAUCGGAGUUG 302

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1. A method of diagnosing or prognosticating a colorectal cancer in asubject, comprising: i) isolating microvesicles from a sample from thesubject; ii) determining a level of at least one miR gene product in themicrovesicles, wherein the at least one miR gene product comprisesmiR-19a, miR-21, miR-127, miR-31, miR-96, miR-135b, miR-183, miR-30c,miR-133a, mir-143, miR-133b and miR-145; and iii) comparing the level ofthe at least one miR gene product in the sample to a control, wherein anincrease in the level of at least one of miR-19a, miR-21, miR-127,miR-31, miR-96, miR-135b and miR-183, and/or a decrease in the level ofat least one of miR-30c, miR-133a, mir-143, miR-133b and miR-145, in thesample from the subject, relative to that of the control, is diagnosticor prognostic of the colorectal cancer. 2-33. (canceled)
 34. The methodof claim 1, wherein the control is selected from the group consistingof: i) a reference standard; ii) the level of the at least one miR geneproduct from a subject that does not have colorectal cancer; and iii)the level of the at least one miR gene product from a sample of thesubject that does not exhibit colorectal cancer.
 35. The method of claim1, wherein the subject is a human. 36-44. (canceled)
 45. The method ofclaim 1, wherein determining the level of at least one miR gene productcomprises: (a) labeling the miR isolated from a the sample from thesubject; (b) hybridizing the miR to an miR array; and (c) determiningmiR hybridization to the array.
 46. The method of claim 1, whereinidentifying miR differentially expressed comprises generating an miRprofile for the sample and evaluating the miR profile to determinewhether miR in the sample are differentially expressed compared to anormal sample. 47-50. (canceled)
 51. The method of claim 1, wherein thesample comprises a peripheral fluid.
 52. The method of claim 51, whereinthe peripheral fluid comprises blood or a fraction thereof.
 53. Themethod of claim 1, wherein determining the level of at least one miRgene product comprises using real-time PCR.
 54. The method of claim 1,wherein the microvesicles are isolated from the sample usingultracentrifugation and/or flow cytometry.